Doud S K, Chou M M, Kendall D A
Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269.
Biochemistry. 1993 Feb 9;32(5):1251-6. doi: 10.1021/bi00056a008.
A systematic series of mutants has been generated which provides a means for titrating the dependence of protein transport activity on signal peptide hydrophobicity. These mutants involve replacement of the hydrophobic core segment of the Escherichia coli alkaline phosphatase signal peptide while maintaining the natural amino- and carboxyl-terminal segments and the overall length. The new core regions vary in composition from 10:0 to 0:10 in the ratio of alanine to leucine residues. Thus, a nonfunctional polyalanine-containing signal peptide is titrated with the more hydrophobic residue, leucine. Using precursor processing to quantify transport activity, we observe a clear, nonlinear dependence on hydrophobicity. At ratios of alanine to leucine of less than or equal to 8:2, the signal peptide is essentially nonfunctional; at ratios greater than or equal to 3:7, the signal peptide functions efficiently. The midpoint is between alanine to leucine ratios of 6:4 and 5:5. Signal peptides with hydrophobicity just below the midpoint show substantial, additional precursor processing over time while the others do not. The data are consistent with a simple model involving a two-state equilibrium between the untransported and transported species and a change in the delta G of -0.85 kcal/mol for every alanine to leucine conversion.
已生成一系列系统的突变体，这为测定蛋白质转运活性对信号肽疏水性的依赖性提供了一种方法。这些突变体涉及替换大肠杆菌碱性磷酸酶信号肽的疏水核心区段，同时保留天然的氨基和羧基末端区段以及全长。新的核心区域中丙氨酸与亮氨酸残基的比例从10:0到0:10不等。因此，用疏水性更强的残基亮氨酸对无功能的含多聚丙氨酸信号肽进行滴定。利用前体加工来量化转运活性，我们观察到对疏水性有明显的非线性依赖性。当丙氨酸与亮氨酸的比例小于或等于8:2时，信号肽基本上无功能；当比例大于或等于3:7时，信号肽能有效发挥功能。中点在丙氨酸与亮氨酸比例为6:4和5:5之间。疏水性略低于中点的信号肽随着时间的推移会有大量额外的前体加工，而其他信号肽则不会。这些数据与一个简单模型相符，该模型涉及未转运和已转运物种之间的二态平衡，并且每进行一次丙氨酸到亮氨酸的转换，ΔG变化为 -0.85千卡/摩尔。
